Hypoxia induces [Ca2+]I increase and associated vasoconstriction in pulmonary artery smooth muscle cells (PASMCs), but not in systemic artery myocytes. Futhermore, hypoxic Ca2+ and contractile responses are much greater in resistance than conduit PASMCs. However, very little is known about the cellular and molecular mechanisms underlying these functional differences. We and other investigators have consistently demonstrated that ryanodine receptor (RyR) Ca2+ release is critical for hypoxic [Ca2+]I increase and associated vasoconstriction in PASMCs. Our preliminary studies, together with previous findings, suggest that specific RyR subtypes (RyR1, RyR2 and RyR3) are differentially expressed in the vasculature. This heterogeneous nature of expression may contribute to observed differences in excitation-contraction coupling and hypoxic Ca2+ and contractile responses in various blood vessels. In addition, specific interactions of endogenous regulatory molecules FKBP12.6 (RyR inhibitor) and cADPR (RyR activator with distinct RyR subtypes may also play an important role in the heterogeneity of physiological and hypoxic Ca2+ release and vasoconstriction. To address these hypotheses, this proposal seeks to examine the following three questions (Specific Aims): Are RyR subtypes heterogeneously expressed in resistance and conduit pulmonary and mesenteric artery SMCs? 2) Is RyR Ca2+ release heterogeneous in resistance and conduit pulmonary and mesenteric artery SMCs? 3) Do the heterogeneities of RyR subtypes expression and RyR coupling to FKBP12.6 and cADPR explain differences in hypoxic Ca2+ release between resistance and conduit pulmonary and mesenteric artery SMCs? These aims will be pursued by using the state-of-the-art biophysical (confocal microscopy, patch clamp, etc), molecular and genetic approaches (gene knockout and over-expression). Thus, the findings from this proposal will extend our understanding of the cellular and molecular mechanisms that contribute to the differences and heterogeneity of physiological and hypoxic Ca2+ and contractile responses, and may identify novel therapeutic targets for pulmonary hypertension. The proposed studies are also of fundamental physiological significance with respect to the regulation of Ca2+ release in other cell types.